The Copepod Acartia Tonsa Dana in a Microtidal Mediterranean Lagoon: History of a Successful Invasion

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Article The Copepod Acartia tonsa Dana in a Microtidal Mediterranean Lagoon: History of a Successful Invasion

Elisa Camatti *, Marco Pansera and Alessandro Bergamasco

Consiglio Nazionale delle Ricerche, Istituto di Scienze Marine (CNR ISMAR), Arsenale Tesa 104, Castello 2737/F, 30122 Venezia, Italy; [email protected] (M.P.); [email protected] (A.B.) * Correspondence: [email protected]; Tel.: +39-041-2407-978

Received: 13 May 2019; Accepted: 5 June 2019; Published: 8 June 2019

Abstract: The Lagoon of Venicehas been recognized as a hot spot for the introduction of nonindigenous species. Several anthropogenic factors as well as environmental stressors concurred to make this ecosystem ideal for invasion. Given the zooplankton ecological relevance related to the role in the marine trophic network, changes in the community have implications for environmental management and ecosystem services. This work aims to depict the relevant steps of the history of invasion of the copepod Acartia tonsa in the Venice lagoon, providing a recent picture of its distribution, mainly compared to congeneric residents. In this work, four datasets of mesozooplankton were examined. The four datasets covered a period from 1975 to 2017 and were used to investigate temporal trends as well as the changes in coexistence patterns among the Acartia species before and after A. tonsa settlement. Spatial distribution of A. tonsa was found to be significantly associated with temperature, phytoplankton, particulate organic carbon (POC), chlorophyll a, and counter gradient of salinity, confirming that A. tonsa is an opportunistic tolerant species. As for previously dominant species, Paracartia latisetosa almost disappeared, and Acartia margalefi was not completely excluded. In 2014–2017, A. tonsa was found to be the dominant Acartia species in the lagoon.

Keywords: Acartia tonsa; Lagoon of Venice; nonindigenous species; zooplankton distribution; coexistence patterns; niche overlaps; long-term ecological research

1. Introduction Alien invasive species, also called non-native or nonindigenous species (NIS), together with marine pollution, overexploitation of living resources, and physical alteration of habitats, represent the main threats to the world’s oceans on local, regional, and global scales [1]. The planktonic crustacean Acartia tonsa Dana (1849) (calanoid copepod) is an NIS recently introduced in the Mediterranean Sea [2]. Several studies have shown that Acartia NIS are colonizing coastal areas and estuaries by propagation or introduction (e.g., [3,4]). The ability of Acartiidae to cross geographic barriers relies mainly in their capability of producing resting stages [5]. These NIS are modifying the status of native species, which are subject to competitive pressure [6]. A. tonsa is widely distributed in estuarine environments along the Atlantic coasts of North and South America [7] and the Pacific coast of North America [8] where it is the most abundant species. It appeared in the European coasts in the first half of the 20th century, possibly transferred by ship ballast waters [9,10]. In the Mediterranean Sea, A. tonsa was first reported in 1985 in the Etang de Berre, a eutrophic lagoon near Marseilles, France [2]. Only after 1985 was the presence of A. tonsa confirmed in several Italian transitional waters such as in a lagoon of the Po River Delta (northern Adriatic Sea) [11], in the Lagoon of Lesina [12], and in the Lagoon of Venice [13]. In estuarine ecosystems, A. tonsa generally reaches relatively high abundances and becomes often the

Water 2019, 11, 1200; doi:10.3390/w11061200 www.mdpi.com/journal/water Water 2019, 11, 1200 2 of 23 dominant zooplankton species during summer [14], provided that high concentrations of particulate organic carbon and particulate organic matter are available. In fact, the life cycle of this copepod is strictly dependent on the quantity of the available food—the larger the trophic supplies are, the more accelerated its growth rate is [15–17]. The Lagoon of Venice, a large Mediterranean lagoon located in the northwest coast of the Adriatic Sea, presents marked habitat heterogeneity, and the classification of its habitats is still a matter of debate [18,19]. The lagoon is considered the main hotspot for invasive species in the whole Italian coast with the presence of more than 60 NIS, including 29 invertebrates and 34 macrophyte species among which A. tonsa also appears [20–25]. The lagoon is also part of the Long-Term Ecological Research (LTER) network (http://www.lteritalia.it), a global network of research sites located in a wide array of ecosystems. LTER research is a fundamental tool for monitoring environmental changes over time. Zooplankton communities exhibit an intrinsically high variability in transitional environments, and the elucidation of coexistence patterns is a critical question in ecology as well as in accounting for differences in abundances among species. In addition to zooplankton ecological relevance related to the role in the marine trophic network, changes in the community have implications for environmental management and ecosystem services. This work describes relevant steps in the history of invasion and establishment of A. tonsa in the Lagoon of Venice with respect to local stressors. This work provides a recent picture of its distribution along gradients of environmental parameters and identifying the relevant biogeochemical quantities assisting or limiting the successful colonization of the species, mainly compared to congeneric residents in the lagoon (Acartia margalefi Alcaraz (1976), Paracartia latisetosa Krichagin (1873), and Acartia clausi Giesbrecht (1889)). The study will focus on abundance, distribution, and niche interactions of the Acartiidae community to address the question of niche separation and to investigate multidimensional niche breadths under a temporal and trophic gradient. The work aims to identify which ecological factors are most important for A. tonsa population structure and organization and to provide a possible key to disentangle the roles of Acartia lagoon dominant species based on their niche characteristics. Identification or exclusion of possible overlaps among realized niches in the space of considered environmental variables will aim to highlight the specialization of each species and the relevance of coexistence mechanisms in the structuration of the community. Combining spatial and temporal frames in the three different periods taken into consideration, along a gradient from the mainland to the inlets, determining species’ relative abundance distributions (RADs) in a given habitat and time will support the comparison of the reciprocal position of the species of interest within the mesozooplankton community and also how they eventually interacted in a competitive way.

2. Materials and Methods

2.1. Study Area The Lagoon of Venice is a large Mediterranean lagoon with an area of approximately 500 km2 stretching along the northwest coast of the Adriatic Sea (Figure1). Three inlets connect the lagoon to the sea, and its general circulation results from the superposition of tide, wind, and topographic control [26,27]. The eﬀective renewal rate of water is on the order of a few days for the areas closest to the inlets and up to a month for the innermost areas [26,28]. Its depth is very shallow, some 1.3 m on average and more than 10 m in the deepest channels connecting the three inlets with the city of Venice and with the industrial area of Marghera. The Lagoon of Venice is surrounded by urban and industrial areas. Moreover, ﬁshery, mariculture, and tourist activities are very developed. Because of the complex interplay of the variety of simultaneously occurring stressors, the lagoon experiences high variability in most of the environmental parameters, showing high habitat heterogeneity [29,30]. Water 2019, 11, 1200 3 of 23 Water 2019, 11, x FOR PEER REVIEW 3 of 24

Figure 1.1. StudyStudy areaarea (Lagoon (Lagoon of of Venice, Venice, Italy) Italy) and and location location of sampling of sampling stations stations during during 1975–1980 1975– (grey1980 (greytriangles, triangles including, including Lido), Lido), 1997–2002 1997– (black2002 (black circles), circles), 2003–2004 2003 (grey–2004 squares),(grey square ands 2014–2017), and 2014 (black–2017 (blacktriangles, triangles including, including San Giuliano, San Giuliano, Marghera, Marghera, Fusina, Fusina, and Lido). and Lido). 2.2. Zooplankton and Environmental Variables Datasets 2.2. Zooplankton and Environmental Variables Datasets Four zooplankton datasets (DSs) from surveys carried out in the Lagoon of Venice were considered Four zooplankton datasets (DSs) from surveys carried out in the Lagoon of Venice were in this study: the first dataset refers to the situation before the settlement of A. tonsa (DS1: period considered in this study: the first dataset refers to the situation before the settlement of A. tonsa (DS1: 1975–1980, monthly sampling) and the three after the first record A. tonsa in the lagoon (DS2: period period 1975–1980, monthly sampling) and the three after the first record A. tonsa in the lagoon (DS2: 1997–2002, monthly sampling; DS3: period 2003–2004, monthly sampling; and DS4: period 2014–2017, period 1997–2002, monthly sampling; DS3: period 2003–2004, monthly sampling; and DS4: period seasonal sampling). 2014–2017, seasonal sampling). The first dataset (DS1), concerning data prior to the establishment of A. tonsa, was used for RAD The first dataset (DS1), concerning data prior to the establishment of A. tonsa, was used for RAD analysis to compare the different relative species’ abundance distributions as a function of the presence analysis to compare the different relative species’ abundance distributions as a function of the of A. tonsa along an expected environmental gradient from the sea to the mainland (Lido inlet, Crevan, presence of A. tonsa along an expected environmental gradient from the sea to the mainland (Lido Dese; Figure1). The second dataset (DS2), the most evenly distributed in terms of spatial and temporal inlet, Crevan, Dese; Figure 1). The second dataset (DS2), the most evenly distributed in terms of coverage, was used to investigate patterns and trends of A. tonsa almost immediately after its first spatial and temporal coverage, was used to investigate patterns and trends of A. tonsa almost record in an intermediate area of the lagoon, called Palude della Rosa. In this case, zooplankton immediately after its first record in an intermediate area of the lagoon, called Palude della Rosa. In samplings were collected from May 1997 to April 2002 at five stations located in the northern part this case, zooplankton samplings were collected from May 1997 to April 2002 at five stations located of the lagoon (San Giuliano, Marghera, Fusina, Lido, and Palude della Rosa; Figure1) characterized in the northern part of the lagoon (San Giuliano, Marghera, Fusina, Lido, and Palude della Rosa; by a complex interplay of freshwater and marine inputs [26,28] and by anthropogenic pressure [31]. Figure 1) characterized by a complex interplay of freshwater and marine inputs [26,28] and by San Giuliano collects urban waste water from the town of Mestre, where phytoplankton blooms often anthropogenic pressure [31]. San Giuliano collects urban waste water from the town of Mestre, where develop [32,33]. Marghera is influenced by industrial pollution. Fusina is affected by thermal pollution phytoplankton blooms often develop [32,33]. Marghera is influenced by industrial pollution. Fusina from a thermo-electric power plant. Lido, in the northernmost inlet of the lagoon, is characterized by is affected by thermal pollution from a thermo-electric power plant. Lido, in the northernmost inlet substantial lagoon shelf exchanges. Palude della Rosa is a typical lagoon environment, influenced both of the lagoon, is characterized by substantial lagoon shelf exchanges. Palude della Rosa is a typical by freshwater and, to a lesser extent, by shelf water transported this far by tides. lagoon environment, influenced both by freshwater and, to a lesser extent, by shelf water transported this far by tides.

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The recent spatial distribution of A. tonsa populations in the lagoon, and its potential diverse response in areas subjected to different natural and anthropogenic influences, were investigated through the analysis of samples derived from monthly samplings, from April 2003 to March 2004, at six stations inside the lagoon and one station in the Adriatic Sea site just outside the Lido inlet (third data set DS3, Figure1). Thus, with respect to the 1997–2002 stations, this dataset provided more coverage and representativeness of different areas in the lagoon with different natural and anthropogenic stressors, including a station on the outer shelf. Station 1M (Figure1) was chosen to verify the presence of the species in the coastal sea, whereas the other stations were selected based on the following rationale: station 2B was influenced by freshwaters; station 7B collected urban waste; station 9B was affected by thermal pollution; station 11B was located in an intermediate area between northern and central lagoon basins; station 14B was in a site with seagrass meadow; and station 17B was located in the southern lagoon basin (Figure1). Station 1M was not sampled in September 2003 and February 2004, and station 17B was not sampled in April 2003. All samples reported abundances of phytoplankton and mesozooplankton organisms as well as physical and chemical parameters related to water quality, in particular: temperature, salinity, dissolved oxygen, chlorophyll a, and particulate organic carbon (POC). Samples related to the most recent period 2014–2017 (DS4) were used to confirm the presence and distribution of A. tonsa ten years after the previous survey along a gradient from the inner lagoon to the outer shelf. In this case, eight stations were sampled. The Inside station (Figure1) was located in the inner channel inside the industrial harbour of Marghera, Port1 was in the lee of the cruise ships docks, 7M was at the yachting mooring docks, and PTF was the CNR Acqua Alta Oceanographic tower (http://www.ismar.cnr.it/infrastructures/piattaforma-acqua-alta), situated few miles offshore the lagoon in the Northern Adriatic. The remaining four stations corresponded to Marghera, Fusina, San Giuliano, and Lido sites of the period 1997–2002. All the considered 65 samples reported abundances of mesozooplankton organisms (66 present taxa) as well as physical and chemical parameters related to water quality, in particular: temperature, salinity, dissolved oxygen, chlorophyll a, nutrients, and Secchi disk. Sampling procedures and analyses were performed in the same way for all datasets; thus, data from different years and projects were homogeneous and comparable with each other. Zooplankton samples were collected by a plankton horizontal sampler (200 µm mesh net size) and preserved with borax buffered formaldehyde for microscopic analysis. Taxonomic and quantitative zooplankton determinations were performed using a Zeiss stereomicroscope at the lowest possible taxonomic level (species for copepods and cladocerans). Each sample was poured into a beaker with 200 cm3 of filtered seawater to allow a thorough mixing for random distribution of the organisms. At least four aliquots of the samples were analysed while the entire sample was checked against the identification of rare species [34,35]. Phytoplankton samples resulting from the DS3 were collected in 250 cm3 dark glass bottles and fixed with 10 cm3 of 20% hexamethylentetramine buffered formaldehyde [36]. Counts were taken according to Utermöhl’s (1958) [37] method, using an inverted microscope (Zeiss Axiovert 35) after 2 to 10 cm3 subsamples had settled for 24 h [38]. Temperature, salinity, and dissolved oxygen were measured in situ using a CTD (Conductivity Temperature Depth) Idronaut Ocean Seven 316 Multiprobe. Particulate organic carbon (POC) was determined using a CHN (Carbon Hydrogen Nitrogen) analyser [39], whereas surface chlorophyll a (Chl a) was analysed with spectrophotometric methods [40].

2.3. Statistical Analyses In order to characterize and explain the A. tonsa population dynamics and trends after its arrival in the lagoon, a seasonal Kendall test (SK) [41] was performed over the data set 1997–2002 (DS2). The test performed the Mann–Kendall (MK) trend test for individual seasons of the year, where season was defined by the user (here, corresponding to monthly values). It then combined the individual Water 2019, 11, 1200 5 of 23 results into an overall test to depict whether the dependent variable changed in a consistent direction (monotonic trend) over time or not. To ascertain if species abundances and environmental parameters were associated, the nonparametric Kendall rank correlation test was carried out [42] on the 2003–2004 dataset (DS3). A correspondence analysis (CA) [43] was carried out on this dataset to highlight the possible relationships and groupings between stations (representative of different habitats) and zooplankton species (proxy of different water masses). CA is the most suitable statistical technique to analyse enumerative data [44]. As it is based on the chi squared metric, this algorithm automatically weights both low and high frequencies. The dataset was organized in a species⁄station matrix. Statistical analyses were performed using MATLAB® software. Species RADs were used to support the comparison of the whole zooplankton community throughout the four decades and the different habitats covered by the available datasets. In particular, three types of habitats were identified along a gradient from the mainland to the sea: the innermost shallow zones characterized by low water circulation (inner), the inlet areas at the interface between the lagoon and the Adriatic Sea with strong marine characters (inlet), and the transition areas with less direct links to the sea (intermediate). For these three habitats, all available samples of each of the three decades (decade D1: 1975–1985, from data set DS1; decade D3: 1995–2005, from data set DS3; recent period D4: 2005–2017, from DS4) were integrated to obtain 3 3 snapshots (before × invasion, early settlement, and more mature condition in each habitat) of the RADs of the overall zooplankton community. Relationships between environmental variables and zooplankton community composition in the current decade (D4)—with particular focus on A. tonsa and the congeneric A. margalefi, P. latisetosa, and A. clausi—were studied using an outlying mean index (OMI) analysis through the ‘ade4’ package in R, extended to the overall zooplankton taxa (dataset DS4). OMI analysis [45] is a multivariate technique to perform niche analyses of species assemblages and explore the relationships between environmental gradients and community structure. The focus is on a specialization criterion called OMI index (i.e., species marginality), which measures the distance between the average habitat conditions used by each species and the average habitat conditions of the sampling area. To characterize the realized niche of each species, the analysis extracts two other terms: the tolerance index (Tol), which measures the habitat breadth of the species, and the residual tolerance (RTol), which represents the variance in the species niche not explained by the measured environmental variables.

3. Results During the decade before the first record of A. tonsa in lagoon (DS1), the mesozooplankton community was composed of more than 40 taxa, with a weak decreasing gradient of overall richness from the inlet (46 taxa) to the intermediate areas (43 taxa) and to the inner zone (41 taxa). Among them, copepods were represented by 24 species, and Acartiidae largely dominated the community with A. clausi being the most abundant taxon in every habitat. The species A. tonsa was not present in any sample of DS1, even in the innermost areas, where the three dominant species (A. clausi, A. margalefi, P. latisetosa) represented more than 80% of the overall community. Conversely, over the analysed 1997–2002 period (DS2), A. tonsa reached higher abundances at the inner lagoon stations San Giuliano (Figure2) and Palude della Rosa (Figure2) and constituted about 90% of the lagoon mesozooplankton community (Figure3). Seasonal cycles (Figure2) showed that the species was, generally, nearly absent in the cold season while annual peaks in abundances were reached in summer. The population started to rapidly increase in May, when suitable water temperatures (>15 ◦C) assisted the growth, and decreased in fall. Annual maxima were reached in different months, depending mainly on the station features. San Giuliano and Palude della Rosa, the stations with larger abundances, had annual peaks in July. Water 2019, 11, 1200 6 of 24 Water 2019, 11, 1200 6 of 23

Figure 2. Box plot of monthly A. tonsa abundance from the dataset 1997–2002 for the ﬁve sampled stations (San Giuliano, Marghera, Fusina, Lido, and Palude della Rosa). The cross (µ) indicates the Figure 2. Box plot of monthly A. tonsa abundance from the dataset 1997–2002 for the five sampled average, 25–75% indicates the interquartile range, and 10–90% indicates the interdecile range. stations (San Giuliano, Marghera, Fusina, Lido, and Palude della Rosa). The cross (μ) indicates the average, 25%–75% indicates the interquartile range, and 10%–90% indicates the interdecile range.

Water 2019, 11, 1200 7 of 24 Water 2019, 11, 1200 7 of 23

Figure 3. Relative abundance, station by station, of A. tonsa with respect to the other Acartia species andFigure the remaining3. Relative zooplanktonabundance, station during by the station, 1997–2002 of A. period. tonsa with respect to the other Acartia species and the remaining zooplankton during the 1997–2002 period. SK analysis showed a statistically significant increase of the species only at the San Giuliano station (TableSK1) along analysis with showed total zooplankton a statistically abundance. significant The increase positive of trendthe species of total only zooplankton at the San appeared Giuliano tostation be due (Table to A. 1) tonsa alongexclusively. with total Atzooplankton Palude della abundance. Rosa, the The second positive station trend in orderof total of zooplankton abundance, theappeared trend of toA. be tonsa dueand to A. total tonsa zooplankton exclusively. abundance At Palude was della also Rosa positive,, the second although station not statistically in order of significantabundance, (Table the 1trend). In theof A. other tonsa stations, and total where zooplanktonA. tonsa abundance abundance was was generally also positive, lower andalthough reached not thestatistically minimum significant at Lido station, (Table the 1). trendIn the wasother found stations, to be where negative A. tonsa and notabundance significant was (Table generally1). lower and reached the minimum at Lido station, the trend was found to be negative and not significant (Table 1). Table 1. Results of the seasonal Kendall test (1997–2002 period). Total Acartia clausi Acartia margalefi Acartia tonsa Table 1. Results of the seasonal Kendall test (1997–2002 period). Zooplankton Tau Acartia0.121 Acartia0.577 Acartia 0.243 Total 0.300 San Giuliano − − pclausi 0.311 margalefi 0.000 tonsa 0.040 Zooplankton 0.011 Tau 0.125 0.687 0.009 0.008 SanFusina Tau −−0.121 −0.577− 0.243− −0.300 p 0.303 0.000 0.943 0.943 Giuliano p 0.311 0.000 0.040 0.011 Tau 0.346 0.728 0.153 0.142 Marghera Tau −−0.125 −0.687− −0.009− −−0.008 Fusina p 0.003 0.000 0.199 0.228 p 0.303 0.000 0.943 0.943 Tau 0.144 0.540 0.094 0.083 Lido Tau −−0.346 −0.728− −0.153− −−0.142 Marghera p 0.225 0.003 0.430 0.480 p 0.003 0.000 0.199 0.228 Palude della Tau 0.059 0.688 0.078 0.067 Tau −−0.144 −0.540− −0.094 −0.083 LidoRosa p 0.620 0.000 0.514 0.572 p 0.225 0.003 0.430 0.480 Palude Tau −0.059 −0.688 0.078 0.067 dellaDespite Rosa the positive,p significant0.620 trend of A. tonsa0.000at San Giuliano,0.514 the estimate of Sen’s0.572 slope [46] was 30 ind (number of individuals)/m3/year. This negligible increase over time (Figure4) may be suggestiveDespite of the a steady positive, state significant of the population trend of A. (i.e., tonsa a matureat San Giuliano, stage of colonization).the estimate of AlongsideSen’s slope the[46] significantlywas 30 ind (number increasing of trend individuals)/m of A. tonsa3/year.abundance This negligible at the San increase Giuliano over station, time SK (Figure highlighted 4) may an be oppositesuggestive trend of a for steady the other state representativeof the population species (i.e., of a thematureAcartia stagegenus of colo historicallynization). relevant Alongside in the the lagoon:significantlyA. margalefi increasingand trendA. clausi of A.(Table tonsa1 ).abundance In particular, at theA. San margalefi Giulianosignificantly station, SK (in highlighted a statistical an sense)opposite decreased trend for at allthe stations, other representative providing additional species evidenceof the Acartia of the genus ongoing historically decline in relevant abundance in the of thislagoon: species. A. margalefi and A. clausi (Table 1). In particular, A. margalefi significantly (in a statistical sense) decreased at all stations, providing additional evidence of the ongoing decline in abundance of this species.

Water 2019, 11, 1200 8 of 23 Water 2019, 11, 1200 8 of 24

Figure 4. Time series of monthly A. tonsa abundance (bars) along with a 12-month running mean and medianFigure and 4. Sen’sTime trendseries lineof monthly at the San A. Giulianotonsa abundance station during (bars) along 1997–2002. with a 12-month running mean and median and Sen’s trend line at the San Giuliano station during 1997–2002. Analyses related to the April 2003 to March 2004 period (DS3) allowed characterization of the actual distributionAnalyses related of A. tonsa to theover April an environmental 2003 to March gradient 2004 period in the (DS3) Lagoon allowed of Venice. characterization The presence of of the a decreasingactual distribution gradient ofof salinityA. tonsacan over be an noted, environmental shifting from gradient stations in nearthe Lagoon inlets to of stations Venice. well The insidepresence theof lagoon a decreasing (Table2). gradient Higher mean of salinity salinity can values be noted, were foundshifting at stationfrom stations 14B, which near was inlets located to stations near an well inletinside and, the therefore, lagoon directly (Table 2). inﬂuenced Higher mean by tidal salinity exchanges, values and were the found marine at station coastal 14B, station which 1M (Tablewas located2). Lowernear salinity an inlet values and, therefore, were observed directly at the influenced innermost by stations tidal exchanges, (2B, 7B, and and 9B). the The marine presence coastal of A.station tonsa 1M at station(Table 1M2). Lower was negligible salinity values and exclusively were observed was dueat the to innermost ebb tidal transport,stations (2B, as 7B, this and species 9B). The is usually presence notof resident A. tonsa in at marine station waters. 1M was Indicators negligible of and trophic exclusively waterquality—particulate was due to ebb tidal organic transport, carbon as this (POC), species Chlisa usuallyconcentration, not resident and phytoplankton in marine waters. abundance—showed Indicators of trophic an opposite water quality—particulate gradient with respect organic to salinity,carbon with (POC), the largerChl a valuesconcentration, at stations and 2B, phytoplankton 7B, and 9B (Table abundance—showed2). The species recorded an opposite the largest gradient abundanceswith respect in those to salinity, stations with (2B and the 9B) larger where values POC, at Chl stationsa, and phytoplankton2B, 7B, and 9B values (Table were 2). highThe species and whererecorded also the the mean largest annual abundances mesozooplankton in those stations abundances (2B and were9B) where highest POC, (6386 Chl a10,327, and phytoplankton and 4892 3 ± ± 11,350values ind were m− respectively;high and where Table also2). the Lowest mean abundanceannual mesozooplankton at the site 14B correspondedabundances were to the highest lowest (6386 concentrations± 10,327 and of POC4892 and± 11,350 Chl a asind well m− as3 respectively; low phytoplankton Table abundance2). Lowest (Tableabundance2). at the site 14B correspondedPercentage contributions to the lowest of concentrations the most abundant of POC taxa and to totalChl a zooplankton as well as low community phytoplankton in each abundance station are(Table reported 2). in Table3. A few species, such as the copepods A. tonsa, A. clausi, Paracalanus parvus Claus (1863), and Centropages ponticus Karavaev (1895), were always present in the zooplankton community over the whole investigated area with a percentage contribution greater than 76%. A. tonsa was the most abundant in all lagoon stations with the exception of 14B and 1M stations, which were dominated by A. clausi. The percent contributions of the codominants P. parvus and C. ponticus were generally lower than 5% except for the 14% contribution of P. parvus at site 1M. The cladoceran Penilia avirostris Dana (1849) was present only at the marine coastal station 1M. The other taxa (i.e., decapods larvae) contributed with low abundances to zooplankton composition.

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Table 2. Average and standard deviation values of the hydro-chemical data and biological concentrations (phytoplankton, A. tonsa, and main mesozooplankton groups) at the seven stations for the 2003–2004 period.

1M 2B 7B 9B 11B 14B 17B Stations Avg SD Avg SD Avg SD Avg SD Avg SD Avg SD Avg SD

Temperature (◦C) 16 8 16 9 17 9 18 8 16 10 16 10 16 9 Salinity 35 2 29 4 32 4 32 3 34 3 35 2 33 4 Relative oxygen (%) 96 5 100 9 102 15 90 7 83 19 92 7 95 5 1 POC (µg L− ) 308 73 750 630 747 415 668 386 485 211 345 141 947 886 1 Chlorophyll a (µg L− ) 1.78 0.92 4.40 4.32 7.02 6.21 7.81 12.69 2.09 1.46 1.54 0.55 3.84 4.52 1 Total phytoplankton (cell L− ) 2541 3176 10,831 16,019 4382 4592 5716 6874 2082 2406 2128 1993 8318 15,745 3 Acartia tonsa (ind m− ) 23 69 5998 10,333 976 1658 4456 11,209 2823 4986 75 192 1234 2744 3 Other copepods (ind m− ) 3702 5494 316 415 521 1015 257 312 602 588 588 1170 144 167 3 Cladocerans (ind m− ) 186 328 2 5 8 26 4 10 9 14 8 22 0 1 3 Other zooplankton (ind m− ) 142 115 69 110 136 290 176 244 161 272 56 54 42 53 3 Total zooplankton (ind m− ) 4053 5906 6386 10328 1642 2062 4893 11349 3595 5207 727 1233 1419 2781 Water 2019, 11, 1200 10 of 24

Table 3. Mean percentage contributions of the most abundant species/taxa at the seven stations for the 2003–2004 period.

1M 2B 7B 9B 11B 14B 17B Acartia clausi 69 1 21 1 8 59 4 Acartia tonsa 1 94 59 91 79 10 87 Centropages ponticus 4 1 2 1 2 2 1 Corycaeus spp. 1 Harpacticoid spp. 3 2 1 Oithona similis 1 6 Oncaea spp. 2 Paracalanus parvus 14 2 4 2 5 5 2 Pseudocalanus elongatus 1 1 1 1 Penilia avirostris 1 Podon polyphemoides 3 Chaetognaths 1 Cirriped larvae 1 1 Decapod larvae 6 2 3 1 1 Fish eggs 1 1 Water 2019, 11, 1200 Gastropod larvae 2 10 of 23

WithWith respect respect to to the the entire entire zooplankton zooplankton community community,, total total abundances abundances increased increased in in late late spring spring 3 andand summer summer at all at stations all stations except except 1M where, 1M where, instead, instead, spring maxima spring maximawere observed were observedin May (17 in × 10 May −3 3 −3 ind(17 m 10) and3 ind June m 3(8) and× 10 June ind m (8 ) mainly103 ind because m 3) mainly of the becausecopepod ofA. theclausi copepod (FigureA. 5). clausi Similar(Figure to the5 ). × − × − datasetsSimilar 1997–2002 to the datasets (DS2), 1997–2002 the warm (DS2), season the corresponded warm season correspondedto the maxima to in the abundances maxima in of abundances A. tonsa. Inof general,A. tonsa the. In main general, mesozooplankton the main mesozooplankton groups showed groups seasonal showed fluctuations seasonal ﬂuctuations with maxima with during maxima warmduring periods; warm copepods periods; dominated copepods dominatedthroughout throughoutthe year. A. themargalefi year. showedA. margaleﬁ very lowshowed abundances very low (maximum mean value of 10 ind m−3 in June), while3 P. latisetosa was never found. The rest of the abundances (maximum mean value of 10 ind m− in June), while P. latisetosa was never found. The rest zooplanktonof the zooplankton community community was mainly was mainly represented represented by the by mero the meroplankton,plankton, especially especially by bydecapod, decapod, gastropod,gastropod, and and cirriped cirriped larvae larvae during during the the spring spring and and summer summer periods periods (Figure (Figure 5).5).

Water 2019, 11, 1200 11 of 24

Figure 5. Monthly abundance of A. tonsa with respect to other Acartia species and remaining zooplankton Figureat coastal 5. Monthly station 1Mabundance (top panel of) andA. attonsa lagoon with stations respect (bottom to other panel Acartia) for thespecies 2003–2004 and remaining period. zooplankton at coastal station 1M (top panel) and at lagoon stations (bottom panel) for the 2003–2004 A match between abundances and distribution of A. tonsa and environmental parameters emerged period. from the Kendall rank correlation test for DS3 (Table4), which showed that A. tonsa was the only speciesA match of dominant between copepods abundances positively and anddistribution significantly of correlatedA. tonsa and with environmental temperature, phytoplankton, parameters emergedPOC and from Chl the a concentrations,Kendall rank correlation as well as testA. formargalefi DS3 (Tablespecies. 4), which However, showed the positivethat A. tonsa correlation was the of onlyA. margalefispecies withof dominant POC was notcopepods significant. positively With respect and significantly to the same parameters,correlated thewith congener temperature,A. clausi phytoplankton,showed the opposite POC and correlation Chl a concentrations, and was not statisticallyas well as A significant.. margalefi species. However, the positive correlation of A. margalefi with POC was not significant. With respect to the same parameters, the congener A. clausi showed the opposite correlation and was not statistically significant.

Table 4. Results of the Kendall rank correlation test at seven stations for the 2003–2004 period. Statistically significant values are marked in bold.

Dissolved Particulate Inorganic Chlorophyll Temperature Salinity Oxygen Organic Phytoplankton Nitrogen a Carbon (POC) (DIN) (°C) (%) (µg L−1) (µg L−1) (µg L−1) (cell L−1) Acartia clausi −0.15 −0.09 0.02 0.09 −0.03 −0.03 −0.10 Acartia 0.38 −0.07 0.09 −0.31 0.31 0.24 0.35 margalefi Acartia tonsa 0.49 0.12 −0.17 −0.48 0.54 0.45 0.62 Centropages 0.49 0.41 −0.25 −0.54 0.35 0.25 0.44 ponticus Corycaeus spp. −0.54 −0.05 0.14 0.39 −0.43 −0.37 −0.53 Harpacticoid 0.17 −0.07 0.01 −0.10 0.20 0.23 0.12 spp. Oithona similis −0.43 −0.07 0.20 0.24 −0.30 −0.21 −0.37 Oncaea spp. −0.39 0.05 0.02 0.23 −0.28 −0.31 −0.36 Paracalanus 0.08 0.31 −0.11 −0.19 0.09 0.04 0.07 parvus Pseudocalanus −0.58 −0.15 0.20 0.43 −0.45 −0.33 −0.48 elongatus Penilia 0.19 0.30 −0.25 −0.24 0.14 0.01 0.16 avirostris Podon 0.17 0.06 −0.09 −0.22 0.15 0.01 0.24 polyphemoides Chaetognats 0.13 0.29 −0.15 −0.23 0.11 −0.03 0.15

Water 2019, 11, 1200 11 of 23

Table 3. Mean percentage contributions of the most abundant species/taxa at the seven stations for the 2003–2004 period.

Table 4. Results of the Kendall rank correlation test at seven stations for the 2003–2004 period. Statistically signiﬁcant values are marked in bold.

Dissolved Particulate Inorganic Organic Temperature Salinity Oxygen Chlorophyll a Phytoplankton Nitrogen Carbon (DIN) (POC) 1 1 1 1 (◦C) (%) (µg L− )(µg L− )(µg L− ) (cell L− ) Acartia clausi 0.15 0.09 0.02 0.09 0.03 0.03 0.10 − − − − − Acartia margaleﬁ 0.38 0.07 0.09 0.31 0.31 0.24 0.35 − − Acartia tonsa 0.49 0.12 0.17 0.48 0.54 0.45 0.62 − − Centropages ponticus 0.49 0.41 0.25 0.54 0.35 0.25 0.44 − − Corycaeus spp. 0.54 0.05 0.14 0.39 0.43 0.37 0.53 − − − − − Harpacticoid spp. 0.17 0.07 0.01 0.10 0.20 0.23 0.12 − − Oithona similis 0.43 0.07 0.20 0.24 0.30 0.21 0.37 − − − − − Oncaea spp. 0.39 0.05 0.02 0.23 0.28 0.31 0.36 − − − − Paracalanus parvus 0.08 0.31 0.11 0.19 0.09 0.04 0.07 − − Pseudocalanus elongatus 0.58 0.15 0.20 0.43 0.45 0.33 0.48 − − − − − Penilia avirostris 0.19 0.30 0.25 0.24 0.14 0.01 0.16 − − Podon polyphemoides 0.17 0.06 0.09 0.22 0.15 0.01 0.24 − − Chaetognats 0.13 0.29 0.15 0.23 0.11 0.03 0.15 − − − Cirriped nauplii 0.42 0.10 0.18 0.35 0.41 0.31 0.33 − − Decapod larvae 0.36 0.25 0.20 0.46 0.37 0.30 0.42 − − Fish eggs 0.44 0.24 0.17 0.41 0.39 0.19 0.42 − − Gastropod larvae 0.19 0.21 0.11 0.24 0.13 0.05 0.08 − −

The CA was suggestive of a separation of the stations into one main group, which included lagoon stations 2B, 9B, 17B, 11B, and 7B, and the other two isolated stations located (i) in an area filled by seagrass in the central basin close to an inlet (14B), which was heavily influenced by tidal exchanges, and (ii) the coastal station 1M (Figure6). The main group, associated mainly with inner lagoon sites, included all stations where A. tonsa dominated and where all trophic parameters favored its abundance. These stations had common species compositions because their species percentage distributions were similar with respect to all other stations. In fact, the same species percentage distribution considerably differed at the inlet station 14B and coastal station 1M. Indeed, Figure7 shows that station 14B was characterized by a more heterogeneous community at the taxa level, and that higher percentages of marine taxa prevailed at station 1M such as appendicularians and cladocerans P. avirostris, Evadne spp., and Podon polyphemoides Leuckart (1859). The strictly neritic species A. clausi was found in both stations 1M and 14B, sites with high salinity and low inorganic nutrient waters, confirming the preference Water 2019, 11, 1200 12 of 24

Cirriped 0.42 0.10 −0.18 −0.35 0.41 0.31 0.33 nauplii Decapod 0.36 0.25 −0.20 −0.46 0.37 0.30 0.42 larvae Fish eggs 0.44 0.24 −0.17 −0.41 0.39 0.19 0.42 Gastropod 0.19 0.21 −0.11 −0.24 0.13 0.05 0.08 larvae

The CA was suggestive of a separation of the stations into one main group, which included lagoon stations 2B, 9B, 17B, 11B, and 7B, and the other two isolated stations located (i) in an area filled by seagrass in the central basin close to an inlet (14B), which was heavily influenced by tidal exchanges, and (ii) the coastal station 1M (Figure 6). The main group, associated mainly with inner lagoon sites, included all stations where A. tonsa dominated and where all trophic parameters favored its abundance. These stations had common species compositions because their species percentage distributions were similar with respect to all other stations. In fact, the same species percentage distribution considerably differed at the inlet station 14B and coastal station 1M. Indeed, Figure 7 shows that station 14B was characterized by a more heterogeneous community at the taxa level, and that higher percentages of marine taxa prevailed at station 1M such as appendicularians and cladocerans P. avirostris, Evadne spp., and Podon polyphemoides Leuckart (1859). The strictly neritic Waterspecies2019, 11A., 1200clausi was found in both stations 1M and 14B, sites with high salinity and low inorganic12 of 23 nutrient waters, confirming the preference for coastal waters. Station 7B, the most distant in CA space from the other lagoon stations, was characterized mainly by the presence of A. margalefi, fish larvae, for coastal waters. Station 7B, the most distant in CA space from the other lagoon stations, was and copepod nauplii. characterized mainly by the presence of A. margaleﬁ, ﬁsh larvae, and copepod nauplii.

Figure 6. Results of the correspondence analysis (CA) performed on seven stations and total annual abundancesFigure 6. Results (May 2003–April of the correspondence 2004). The ﬁrst analysis letter (CA) of each performed species’ on name seven corresponds stations and to thetotal actual annual positionabundances on the (May CA space. 2003–April For enhanced 2004). The readability, first letter only of each species species’ with name relative corresponds abundance to> 5%the actualare Water 2019, 11, 1200 13 of 24 shown.position Stations on the B2 CA and space. B9 as wellFor e asnhanced Polychaeta readability, larvae, Decapoda only species larvae, with and relative Cirripeda abundance nauplii species>5‰ are haveshown. been Stations moved from B2 and their B9 respective as well as original Polychae positions,ta larvae, indicated Decapoda by the larvae, black and lines. Cirripeda nauplii species have been moved from their respective original positions, indicated by the black lines.

Figure 7. Relative abundance, station by station, of A. tonsa with respect to the other Acartia species and theFigure remaining 7. Relative zooplankton abundance, during station 2003–2004by station, of period. A. tonsa with respect to the other Acartia species and the remaining zooplankton during 2003–2004 period. Data related to the last period 2014–2017 (DS4), though only seasonal, confirmed the presence and the dominanceData related of to threethe last principal period 2014–2017Acartia species (DS4), though inside only thelagoon: seasonal,A. confirmed margalefi the, A. presence clausi and A. tonsaand. Athe di dominancefferent distribution of three principal of these Acartia species species along inside physical theand lagoon: trophic A. margalefi gradients, A. wasclausi also and found: A. A. margalefitonsa. Acoexisted different distribution with A. tonsa of mainlythese species in the along intermediate physical and lagoon trophic areas gradients (Figure was8). Both also found: virtually disappearedA. margalefi at coexisted stations with A. marine tonsa mainly features in the (Lido intermediate and PTF) lagoon where areasA. (Figure clausi instead 8). Both dominated.virtually disappeared at stations with marine features (Lido and PTF) where A. clausi instead dominated. P. P. latisetosa was found with very limited abundance in the inner lagoon stations (Inside, Marghera, and latisetosa was found with very limited abundance in the inner lagoon stations (Inside, Marghera, and Fusina) only in May 2014 at 4 ind m 3 or less. Fusina) only in May 2014 at 4 ind −m−3 or less.

Water 2019, 11, 1200 13 of 23 Water 2019, 11, 1200 14 of 24

Figure 8.FigureSeasonal 8. Seasonal relative relative abundance abundance of A. of tonsaA. tonsawith with respect respect to to other other Acartia speciesspecies and and remaining remaining zooplanktonzooplankton at stations at stations for the for2014–2017 the 2014–2017 period. period. Data Data related relatedto to 7M,7M, Port1, and and inside inside refer refer only only to to May 2014,May October 2014, October 2014, 2014, and Februaryand February 2015 2015 sampling sampling dates. dates.

RDA analysisRDA analysis showed showed that that the relativethe relative rank rank distribution distribution of of thethe mostmost representative representative zooplankton zooplankton speciesspecies in the communityin the community changed changed over over time time and and in in space space by by comparing comparing the the periods periods before before and and after after the arrivalthe arrival of A. tonsa of A.. tonsa First. First of all, of Figureall, Figure9 shows 9 shows that that much much of of the the RAD RAD shapeshape was was steeply steeply dominated dominated by the arrivalby the arrival of A. tonsaof A. ,tonsa especially, especially in inner in inner areas areas where where the the species species reachedreached maximum maximum abundance abundance values and exceeded those of the congeneric ones. A. clausi maintained the most representative values and exceeded those of the congeneric ones. A. clausi maintained the most representative relative abundance of the Acartia genus throughout the period prior to the arrival of A. tonsa and in all three considered areas (Figure9). It maintained the same relative rank also in the following periods but only Water 2019, 11, 1200 14 of 23 in the inlet areas. In the years following its appearance, A. tonsa replaced A. clausi in the intermediate and inner areas. P. latisetosa reached an important distribution rank only in the internal areas and in the pre-A. tonsa period, after which it could always be found on the low-rank curve tail both in spatial and temporal terms. A. margaleﬁ, also with an important relative abundance, before the arrival of A. tonsa, especially in the most typically lagoon areas (intermediate and inner), fell in rank with the appearance of A. tonsa (D3), but then (D4) recovered in terms of presence and abundance mainly in the intermediate areas (Figure9).

Figure 9. Rank abundance distribution (RAD) and occurrence of the zooplankton community of the Venice lagoon during the period of study (decade D1: 1975–1985, from data set DS1; decade D3: 1995–2005, from data set DS3; and recent period D4: 2005–2017, from data set DS4). The samples were lumped according to the three habitat types along a gradient from the mainland to the sea (see text). In every frame, the upper curve gives the ranked occurrence of each species in the lumped N samples; the Acartia species’s relative ranks are highlighted along the lower RAD curve.

With an OMI analysis, we computed and tested niche parameters (Table5) to describe marginality, tolerance, and, thus, the variability of responses of Acartia species to environmental variables as well as their possible niche overlap. Figure 10 shows the representation of the statistically significant species’ realized niche position of Acartia species on the first factorial plane of the OMI analysis whose origin represents the average habitat conditions of the sampling area. The two first axes of the OMI analysis accounted for 81% of the marginality (69% for the first axis). As a consequence, subsequent graphs used only these two axes. Seventeen out of 66 taxa showed a significant deviation (p < 0.05) of Water 2019, 11, 1200 15 of 23 their niche from the origin (whereas among the Acartiidae, only A. clausi was significant, p < 0.01) suggesting a significant influence of the environmental conditions for a relevant part of the community (Monte-Carlo krandtest, perm = 999). Furthermore, the between-site analysis confirmed that the environmental gradient of sites (sea, inlet, intermediate, and inner) could be discriminated through the inhabiting zooplankton community (Monte-Carlo rtest, perm = 99, p < 0.01). In our case, the tendency of low values of marginality for Acartia species indicated that there was no significant difference between the average overall environmental conditions and those where the species were preferentially found. The niches of the three typical lagoon species, P. latisetosa, A. margalefi, and A. tonsa, gravitated around less salty and more trophic environments and also presented an evident overlap. Furthermore, A. tonsa and A. clausi, the latter having more neritic coastal characteristics, were equidistant from the average environmental conditions. Therefore, this showed a clear affinity, or the opposite, depending on the environment taken into consideration: coastal marine sites for A. clausi (sea and inlet in the chart) and lagoon for A. tonsa (intermediate and inner). In particular, A. tonsa had a low marginality and a high residual tolerance to environmental conditions, whereas A. clausi showed an opposite trend but was still relative low OMI values (Table5). As for the other two typical lagoon species, A. margalefi had the lowest value of OMI, while P. latisetosa had the lowest RTol in comparison to the rest of the Acartia species (Table5). This meant that A. clausi was greatly influenced by environmental conditions, whereas P. latisetosa seemed to poorly depend on unconsidered environmental parameters (e.g., POC). Instead, A. tonsa showed wider potential ecological tolerance. In general, most common habitat conditions covered by the sampling units corresponded to the ubiquitous or generalist species. In contrast, specialists, which deviated from these general habitat conditions, demonstrated high OMI values, as in the case of P. latisetosa with respect to the other two lagoon congeners. Tol values showed a high correlation and dependence of P. latisetosa on environmental variables, while A. tonsa, with its high RTol, showed a greater adaptability to the variations of the ecosystem in which it gravitated. A. margalefi had the lowest OMI and Tol values.

Table 5. Niche parameters of the 20 most abundant zooplankton taxa in the Lagoon of Venice during 2014–2017 (outlying mean index (OMI) analysis). The inertia of each species, OMI, the tolerance index (Tol), and the residual tolerance index (RTol) are indicated. The last column (p) represents the percentage of random permutations (out of 1000) that yielded a higher value than the observed OMI (signiﬁcant cases are in bold, p < 0.05).

Species Inertia OMI Tol RTol p Acartia tonsa 12.76 2.80 1.35 8.61 0.497 Acartia clausi 9.37 3.13 2.08 4.15 0.012 Paracalanus parvus 11.78 3.91 2.58 5.28 0.034 Decapoda larvae 19.07 11.62 5.09 2.36 0.620 Acartia margaleﬁ 6.37 1.66 0.90 3.80 0.880 Podonidae 7.09 2.36 0.30 4.43 0.818 Penilia avirostris 8.46 5.69 0.49 2.28 0.030 Appendicularia 9.84 3.35 2.38 4.09 0.567 Centropages ponticus 8.07 2.95 1.29 3.82 0.214 Ascidiacea larvae 14.44 10.33 1.55 2.56 0.728 Echinodermata larvae 10.35 6.66 0.79 2.91 0.015 Oithona similis 10.68 6.38 0.93 3.36 0.011 Nauplii copepoda 7.95 1.93 0.90 5.12 0.819 Evadne nordmani 7.08 5.52 0.21 1.35 0.849 Harpacticoida 13.32 1.63 2.48 9.22 0.802 Clausocalanus spp. 12.91 8.93 0.84 3.15 0.268 Oncaeidae 13.21 8.44 1.74 3.03 0.055 Bivalvia larvae 12.25 1.72 1.45 9.08 0.690 Polychaeta larvae 9.46 1.77 1.01 6.67 0.214 Paracartia latisetosa 1 9.80 4.41 3.11 2.29 0.910 1 P. latisetosa does not fall within the 20 higher ranks. WaterWater2019 2019, 11, 11, 1200, x FOR PEER REVIEW 168 of of 23 24

d = 2 4 Inlet Inner Intermediate Sea

amar

2 aton s plat

0 x acla

-4 -2 0 2 4

Axis 1

Figure 10. OMI analysis. Realized niche position of Acartia species in the Lagoon (2014–2017 dataset). TheFigure site scores10. OMI are analysis. projected Realized on the niche same position first two of axes Acartia of the species OMI in analysis, the Lagoo andn (2014 the habitat–2017 dataset). type is highlightedThe site scores (Labels: are projected acla = A. clausion the; aton same= A.first tonsa two; plataxes= ofP. the latisetosa OMI ;analysis and amar, and= A. the margalefi habitat). type is highlighted (Labels: acla = A. clausi; aton = A. tonsa; plat = P. latisetosa; and amar = A. margalefi). 4. Discussion 4. DiscussionThe occurrence of NIS is increasing in marine and estuarine systems. Among the 955 new taxa reported for the Mediterranean Sea, 42 are planktonic copepods [47]. Their invasive behaviour has been The occurrence of NIS is increasing in marine and estuarine systems. Among the 955 new taxa recognized as one of the major threats to the conservation of the biodiversity and the functioning of reported for the Mediterranean Sea, 42 are planktonic copepods [47]. Their invasive behaviour has marine ecosystems [48–50]. A. tonsa dated its first appearance in the Venice lagoon in 1992. Since then, been recognized as one of the major threats to the conservation of the biodiversity and the functioning itof never marine disappeared, ecosystems and [48 it–50 is] now. A. tonsa present dated throughout its first appearance the year and in dominantthe Venice with lagoon the in exception 1992. Since of colderthen, months.it never The disappeared species is, widespreadand it is now throughout present thethroughout lagoon with the significant year and dominant abundance with except the forexception the areas of closer colder to the months. tidal inlets, The species where the is widespreadtrophic condition throughout is lowered the and lagoon the hydro-chemical with significant A. tonsa characteristicsabundance except become for the less areas favourable closer to to the the tidal species. inlets, In where the Venice the trophic lagoon, condition is islowered currently and consideredthe hydro- achemical stabilized characteristics species. The mostbecome plausible less favourable hypotheses to aboutthe species. the introduction In the Venice of this lagoon, species A. aretonsa that is it was currently brought considered into the outer a stabilized port area species.thanks to The its ability most to plausible produce hypotheses resting eggs, about then via the ballastintroduction water from of this ships species [51–53 are], orthat released it was by brought aquaculture, into the fisheries, outer port or pet area industries thanks to [54 its]. ability to produceOur studyresting showed eggs, then how via the ballast trophic water conditions from ships of [51 the–53] lagoon, or released system by were aquacultur (and stille, are)fisheries the , mainor pet factors industries that influenced[54]. its adaptive success. Unlike the other main species present within the A. tonsa lagoonOur zooplankton study showed communities, how the trophic conditionsseems strongly of the lagoon dependent system on were trophic (and conditions, still are) the as main it is positivelyfactors that correlated influenced with its nutrients, adaptive Chlsuccess.a, POC, Unlike and the phytoplankton other main species concentrations. present within The favourable the lagoon environmentalzooplankton communities, conditions that A. the tonsa species seems found strongly in 1992, dependent fundamentally on trophic characterized conditions, as by it high is positively habitat trophiccorrelated levels with [55], arenutrients, congruent Chl with a, our POC current, and findingsphytoplankton and to the concentrations ecological characteristics. The favourable of the speciesenvironmental [8,15,56]. conditions This allowed that the the settlement species found of A. in tonsa 1992,in fundamentally the Lagoon of characterized Venice. by high habitat trophicMuch levels of the [55] available, are congruent knowledge with about our current zooplankton findings communities and to the ecological of the Venice charact lagoon,eristics mainly of the copepods,species [8,15, derives56]. This from allowed studies the along settlement the physical of A. tonsa and in trophic the Lagoon gradient of Venice. of the northern lagoon. In thisMuch area, Acartiaof the availablegenus usually knowledge represents about a quantitative,zooplankton importantcommunities component of the Venice of the lagoon, zooplankton mainly communities.copepods, derives It shows from spatial studies and along seasonal the physical segregation and trophic patterns gradient associated of the with northern the hydrological lagoon. In conditions,this area, Acartia the seasonal genus variability, usually represents and the trophica quantitative status and, important pollution component of the water of [the31,57 zooplankton,58]. communitiesComparison. It ofshow thes whole spatial zooplankton and seasonal community segregation throughout patterns the associated four decades with andthe thehydrological different habitatsconditions, highlighted the seasonal that variability, in the Venice and lagoon,the trophic before status the and arrival pollution of A. of tonsa the water, the genus[31,57,58]Acartia. dominatedComparison the copepod of the community whole zooplankton with P. latisetosa communityand A. margalefi throughoutusually the found four in decades the innermost and the anddifferent intermediate habitats parts highlighted of the lagoon, that in respectively. the Venice lagoon, In particular, before inthe the arrival more of internal A. tonsa areas,, the genusA. margalefi Acartia anddominatedP. latisetosa theranked copepod second community and third, with respectively, P. latisetosa and in terms A. margalefi of presence usually and found abundance, in the innermost though

Water 2019, 11, 1200 17 of 23

P. latisetosa greatly diminished in significance in the intermediate areas. The dominant species A. clausi ranked always first, mainly in the coastal area. This picture instead has been profoundly modified with the passage from the first analysed decade (i.e., before the invasion of A. tonsa) to the following ones. In fact, the current scenario shows, again, the Acartia species among the dominant species of the entire lagoon zooplankton community, and it is always organized along a spatial gradient determined by salinity and trophic conditions but with modified rankings in the different considered areas. A. clausi no longer dominates all the considered sub-basins, except for the inlet area where it is, again, the most representative species both in space and in time. P. latisetosa, whose presence and dominance were already weak in the intermediate areas, further lost importance in the inner areas and virtually disappeared except in spring 2014. Very little abundance of the species was observed only in the internal channels of the lagoon (Inside, Marghera, and Fusina stations). We argue this could either be due to its segregation in more confined and not yet investigated areas or to the occupation of its original habitat by A. tonsa. In the presence of a large salinity and trophic range, the Acartia species coexist in time with a strong spatial segregation that shows A. clausi limited to the inlet and coastal areas, A. margalefi in the intermediate area, and partly overlapping with A. tonsa that dominates in the innermost areas. During the second decade (1995–2005), coinciding with the first phase of stabilization of the species after its first occurrence dated 1992, A. tonsa seems to clearly supplant the A. clausi and A. margalefi congeners in the intermediate and mostly internal areas, whereas occupation of A. clausi near the lagoon mouths failed. It is interesting to note that, in more recent times (2005–2017), A. margalefi appears to be recovering its original ranking, returning to coexist with A. tonsa in the intermediate areas. As reported in other Adriatic lagoons [59,60], the arrival of A. tonsa may have caused the disappearance or drastic decrease of congeneric species, and the current coexistence of A. tonsa and A. margalefi is probably favored by a greater resilience of the latter as well as a return to more favorable environmental conditions always for the latter (decline in nutrient concentration and increase in ecological status) [61]. Therefore, among the main factors that seem to have favoured the adaptive success of A. tonsa compared to its congeners, we can ascribe the deterioration of the environmental conditions during the 1970s and the 1980s to when the lagoon of Venice was affected by abnormal inputs of pollutants and trophic loadings, mainly phosphorus and nitrogen compounds, which induced massive macroalgae growth (Ulva rigida C. Agardh, 1823) whose decomposition in summer led to frequent dystrophic crises [62–65]. Later, during the 1990s, the Lagoon of Venice experienced significant changes in both primary production and trophic conditions. In particular, Ulva coverage rapidly declined [64], mainly because of climatic changes and the increase of sediment resuspension and sedimentation [66]. As a consequence, since the end of 1980s and through the 1990s, the presence patterns of Acartia species also changed the mesozooplankton composition of the lagoon. Within this scenario, and knowing that A. tonsa can tolerate low dissolved oxygen concentrations and adapt well to hypoxia [67,68], we hypothesized that the hypoxia and hypertrophy in the inner lagoon area could not have hindered the settlement of A. tonsa whose adaptive success almost seemed to have benefited. Our results seem to support the hypothesis that, after a first important collapse of A. margalefi coinciding with the deterioration of environmental conditions and with the advent of A. tonsa, the first remained segregated in the intermediate areas of the lagoon, leaving A. tonsa to the innermost ones. Given the niche overlap that emerged from the OMI analysis, it is quite clear that competition for the same resources may be one of the main factors affecting the coexistence of the different Acartia in the same area, and that the observed decline in abundance of P. latisetosa and A. margalefi could be linked to changes in environmental conditions during the 1980s that favored A. tonsa development. Acartia genus is known to have both herbivorous and omnivorous feeding habits, depending on the environmental resources [69], although little knowledge exists about the nutritional requirements of A. margalefi [70]. The available studies report that P. latisetosa probably has an omnivorous or detritivorous diet just like A. tonsa [71]. Our analyses suggest for these two Water 2019, 11, 1200 18 of 23 congeners a stricter dependence on the considered trophic variables resulting in a stronger competition for food with A. tonsa. Therefore, change in distribution and size of the populations of A. margalefi and P. latisetosa occurred from the second half of the 1980s to the present. This probably depended on the success of recruitment that, in turn, could have been influenced by the system’s trophic conditions but also, consequently, by the presence of effective competitors such as A. tonsa, which are also known to be an active predator of early larval forms. Competition with other copepods, especially congeneric, appears to be the main documented impact of A. tonsa (e.g., [4,51,52,72,73]) besides being known that A. tonsa species may coexist with indigenous Acartiidae (e.g., [6]) or exclude them by competition [5], as suggested in some Mediterranean systems [57,59,60], where it seems that A. margalefi and A. clausi are not able to settle down in large populations in areas colonized by A. tonsa [59,74]. This may indicate that large-scale changes, as well as a biotic interactions with the species and mainly with the “new” species A. tonsa, might be responsible for the evolution in the lagoon of Venice of different dominant copepod associations with respect to the past. The triggering phenomenon seems to have been the anthropic action on such a delicate and dynamic ecosystem, whose impact A. tonsa took advantage of. Currently A. tonsa dominates the intermediate and internal areas of the lagoon, and it reaches the highest population abundance to the detriment of the indigenous congener species. The process, initially favored by the negative effect of changes in environmental conditions, worsened because a clear niche overlap emerged, which was confirmed by our study, in particular between A. tonsa and A. margalefi with A. tonsa benefiting from the point of view of trophic resources. The extent of its area of distribution in the lagoon confirms its euryhaline species characteristics and its excellent adaptability to instantaneous variations in habitat conditions. At present, the lagoon is oligo-mesotrophic over a great part of its surface [61] and dominated by areas classified as polyhaline (18 to 30 salinity) that correspond to the optimal development of A. tonsa (salinity ranging from 15 to 22), where the lowest salinities limit its egg production [75]. The adaptive success of A. tonsa with respect to its congener in the Lagoon of Venice corroborate the findings that A. tonsa is an opportunistic, tolerant species that can take advantage of eutrophic/impacted ecosystems [4,9,31,76,77] and invade them. Restriction of A. tonsa distribution offshore seems mainly influenced by food availability and less by the salinity. In the Lagoon of Venice, A. tonsa can sustain the high energy requirements. Moreover, competition with true marine copepods is reduced because of the habitat selection that takes place along the lagoon–sea gradient. It is also probable that the spatial width and heterogeneity of the Venetian lagoon favored and allowed the gradual recovery of the community of A. margalefi in recent years, which we observed in the present work. Succumbing to the highly competitive level of the alien A. tonsa, A. margalefi seems to have rediscovered a niche that does not completely overlap that of A. tonsa right along the saline and trophic gradient that exists in the lagoon. The reports of new NIS in the Venice lagoon—the copepods Pseudiaptomus marinus Sato (1913), Oithona davisae Ferrari F.D. and Orsi (1984), and ctenophore Mnemiopsis leidyi A. Agassiz (1865) [53,78]—are also very recent. The latter, reported for the first time in the lagoon in 2016, is still present throughout the year, and its strong predator characteristics on the planktonic component [78] could revolutionize again the structure of the Acartia genus and in general of the zooplankton communities in a few years. Copepods are important components of the pelagic food web, as they are themselves food resources for numerous bentho-pelagic invertebrates and planktivorous juveniles of fishes. The advent of new species capable of competing for the same resources, as M. leidyi, could trigger a process at the base of the trophic levels that would affect the highest ones. So, the story of an invasion could therefore not have ended here. Water 2019, 11, 1200 19 of 23

5. Conclusions The Lagoon of Venice has been recognized as a hot spot for the introduction of nonindigenous species [25]. Several anthropogenic factors (industrial and urban pollution, mariculture, shipping, and tourism) as well as environmental stressors (e.g., warming) concurred to make this ecosystem ideal for invasion as in other estuaries and coastal areas of the Mediterranean. In this work, four datasets of mesozooplankton were examined, with particular emphasis on Acartiidae and the alien species A. tonsa. The first dataset, dated 1975–1980, was used as a term of comparison before A. tonsa settlement. The second dataset (during 1997–2002) was used to investigate the seasonal cycle and temporal trends. The third dataset (2003–2004) was used to underpin the spatial variability of abundance and species composition, and the last dataset (the recent period until 2017) was used to confirm the presence of A. tonsa several years later. The selected trend test on A. tonsa abundance did not point out significant overall increases in the period 1997–2002, but it did point out increases in one single station (the most eutrophic one). This may be suggestive that the population has reached a mature stage of colonization in the lagoon. The annual cycle showed maxima in the warm season (mostly July) and negligible abundances in the cold season. Spatial distribution of A. tonsa was found to be significantly, positively associated with temperature, phytoplankton, POC, chlorophyll a, and counter gradient of salinity, which confirmed that A. tonsa was an opportunistic tolerant species that could take advantage of eutrophic ecosystems. This would seem to have occurred at the expense of the previously dominant species A. margalefi and P. latisetosa, which clearly declined in abundance over the last years. While P. latisetosa almost disappeared, A. margalefi was not completely excluded. Stations inside the lagoon showed similar species compositions remarkably different from the station in the outer shelf, which was more representative of coastal conditions and dominated by A. clausi. In 2014–2017, A. tonsa was found to be still one of the dominant Acartia species in the lagoon. Zooplankton is known to be particularly sensitive to environmental changes, whether resulting from natural or anthropogenic forcing [79–82]. The interaction between anthropogenic activity, climate change, and plankton communities, focusing on systematic changes in plankton community structure, abundance, distribution, and phenology over recent decades, is a key global issue as well as the potential socioeconomic impacts of these changes [83]. In environments of relentless evolution such as the Lagoon of Venice, monitoring the ecological dynamics of species is of the utmost importance. In particular, continuous modifications in zooplankton assemblages in response to anthropogenic and environmental stressors must be considered. Ongoing plankton monitoring in the LTER site of the Lagoon of Venice will act as sentinel research to identify future changes in this complex, heavily impacted ecosystem and its related food web.

Author Contributions: E.C. contributed substantially to the study’s conceptualization, data acquisition, analysis, and to the original draft preparation; M.P. contributed to recent data acquisition and analysis and to the review and editing; and A.B. contributed substantially to statistical analyses and to the review of the manuscript. All authors approved the final submitted manuscript. Funding: This work was supported by the LIFE-WATERS Program, EC-sponsored, for the two-year period 1997–1999. The data set 2002–2003 was collected within the framework of the “Integrazione delle Conoscenze del Sistema Ecologico Lagunare (ICSEL)” project, promoted by the Venice Water Authority (Magistrato alle Acque) through Servizio Ambiente—Consorzio Venezia Nuova, while the dataset 2014–2017 derived from the ADRIATIC IPA BALMAS (Ballast Water Management System for Adriatic Sea Protection) Project and from the LTER. Acknowledgments: The authors acknowledge Thetis S.p.A. for providing the environmental data during 2003–2004 period and the CNR colleague F. Acri for his help in carrying out biochemical analyses. We wish to thank F. Bernardi Aubry, M. Bastianini, S. Finotto, A. Schröder, S. Pasqual, L. Dametto, and M. Casula for the fieldwork technical support during sampling and the crew of the M/B “Litus” G. Zennaro, D. Penzo, and M. Penzo. Conflicts of Interest: The authors declare no conflict of interest.

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